As a defense against oxygen radicals, cells possess a series of antioxidant enzymes and low molecular weight antioxidants. Alterations in these regulated reactions cause direct damage to the cell or change the ratios of redox cycling agents. We investigated the effects of (a) enhanced activity of Cu,Zn superoxide dismutase (Cu,ZnSOD) in cell culture and (b) a protective enzyme against thiyl radicals in vitro. NCB-20 cells, a mouse neuroblastoma-Chinese hamster brain hybrid cell line, were transfected with a human pSVneo Cu,ZnSOD gene expression vector. We investigated formation of free radicals in these clones by hydrogen peroxide generated by a glucose and glucose oxidase coupled system. EPR and spin trapping studies showed that glutathionyl radicals were produced in cells. The concentration of the radicals in wild-type cells is two-fold higher than that in the overexpressing clones. This indicates that these clones maintain chronic prooxidant states with decreased concentration of reduced glutathione (GSH) due to, in part, free radical production by Cu,ZnSOD with hydrogen peroxide and GSH as substrates. A protector protein (PP) that had been previously purified and cloned protects other enzymes and proteins against inactivation by thiol-induced oxidants. Studies with point mutants (Cys47S and Cys170S) indicate that Cys47 is required for the protection activity. Thiyl radicals were produced and trapped by a spin trap in solution containing oxygen, Fe(III), and dithiothreitol or GSH. The wild-type PP and Cys170S PP quench the spin adduct signals while Cys47S does not. These results together indicate that PP either scavenges thiyl radicals directly or removes one of their reaction products with oxygen, sulphenyl hydroperoxide.